Reduced levels of mitochondrial DNA increases the toxicity of 9-beta-D-arabinofuranosylguanine (araG)

Incubation of cells with thymidine (dThd) is known to cause dNTP pool imbalance as well as deletions and depletion of the mtDNA. In order to gain further understanding in the events involved in dThd toxicity over time, H9 cells were cultured for 20 months in the presence or absence of 1 micro M dThd. The level of mtDNA was reduced by 90% in the cells grown in dThd as compared to the control cells. The H9/dThd cells also showed a 100-fold increased sensitivity towards the cytotoxicity of the antileukemic compound 9-beta-D-arabinofuranolsylguanine (araG).     

Different motifs regulate trafficking of SorCS1 isoforms

The type I transmembrane protein SorCS1 is a member of the Vps10p-domain receptor family comprised of Sortilin, SorLA and SorCS1, -2 and -3. Current information indicates that Sortilin and SorLA mediate intracellular protein trafficking and sorting, but little is known about the cellular functions of the SorCS subgroup. SorCS1 binds platelet-derived growth factor-BB (PDGF-BB) and is expressed in isoforms differing only in their cytoplasmic domains. Here, we identify two novel isoforms of mouse SorCS1 designated m-SorCS1c and -d. In situ hybridization revealed a combinatorial expression pattern of the variants in brain and embryonic tissues. We demonstrate that among the mouse variants, only SorCS1c mediates internalization and that the highly conserved SorCS1c is internalized through a canonical tyrosine-based motif. In contrast, human SorCS1a, whose cytoplasmic domain is completely different from mouse SorCS1a, is internalized through a DXXLL motif. We report that the human SorCS1a cytoplasmic domain interacts with the αC/σ2 subunits of the adaptor protein (AP)-2 complex, and internalization of human SorCS1a and -c is mediated by AP-2. Our results suggest that the endocytic isoforms target internalized cargo to lysosomes but are not engaged in Golgi–endosomal transport to a significant degree.     

Integron-associated gene cassettes in Halifax Harbour: assessment of a mobile gene pool in marine sediments

The integron/gene cassette systems identified in bacteria comprise a class of genetic elements that allow adaptation by acquisition of gene cassettes. Integron gene cassettes have been shown to facilitate the spread of drug resistance in human pathogens but their role outside a clinical setting has not been explored extensively. We sequenced 2145 integron gene cassettes from four marine sediment samples taken from the vicinity of Halifax Nova Scotia, Canada, increasing the number of gene cassettes obtained from environmental microbial communities by 10-fold. Sequence analyses reveals that the majority of these cassettes encode novel proteins and that this study is consistent with previous claims of high cassette diversity as we estimate a Chao1 diversity index of ∼3000 cassettes from these samples. The functional distribution of environmental cassettes recovered in this study, when compared with that of cassettes from the only other source with significant sampling ( Vibrio genomes) suggests that alternate selection regimes might be acting on these two gene pools. The majority of cassettes recovered in this study encode novel, unknown proteins. In instances where we obtained multiple alleles of a novel protein we demonstrate that non-synonymous versus synonymous substitution rates ratios suggest relaxed selection. Cassette-encoded proteins with known homologues represent a variety of functions and prevalent among these are isochorismatases; proteins involved in iron scavenging. Phylogenetic analysis of these isochorismatases as well as of cassette-encoded acetyltransferases reveals a patchy distribution, suggesting multiple sources for the origin of these cassettes. Finally, the two most environmentally similar sample sites considered in this study display the greatest overlap of cassette types, consistent with the hypothesis that cassette genes encode adaptive proteins.     

Proton-translocating transhydrogenase: an update of unsolved and controversial issues

Proton-translocating transhydrogenases, reducing NADP⁺ by NADH through hydride transfer, are membrane proteins utilizing the electrochemical proton gradient for NADPH generation. The enzymes have important physiological roles in the maintenance of e.g. reduced glutathione, relevant for essentially all cell types. Following X-ray crystallography and structural resolution of the soluble substrate-binding domains, mechanistic aspects of the hydride transfer are beginning to be resolved. However, the structure of the intact enzyme is unknown. Key questions regarding the coupling mechanism, i.e., the mechanism of proton translocation, are addressed using the separately expressed substrate-binding domains. Important aspects are therefore which functions and properties of mainly the soluble NADP(H)-binding domain, but also the NAD(H)-binding domain, are relevant for proton translocation, how the soluble domains communicate with the membrane domain, and the mechanism of proton translocation through the membrane domain.     

HIV-1 with multiple CCR5/CXCR4 chimeric receptor use is predictive of immunological failure in infected children

Background

HIV-1 R5 viruses are characterized by a large phenotypic variation, that is reflected by the mode of coreceptor use. The ability of R5 HIV-1 to infect target cells expressing chimeric receptors between CCR5 and CXCR4 (R5^broad viruses), was shown to correlate with disease stage in HIV-1 infected adults. Here, we ask the question whether phenotypic variation of R5 viruses could play a role also in mother-to-child transmission (MTCT) of HIV-1 and pediatric disease progression.

Methodology/Principal Findings

Viral isolates obtained from a total of 59 HIV-1 seropositive women (24 transmitting and 35 non transmitting) and 28 infected newborn children, were used to infect U87.CD4 cells expressing wild type or six different CCR5/CXCR4 chimeric receptors. HIV-1 isolates obtained from newborn infants had predominantly R5^narrow phenotype (n = 20), but R5^broad and R5X4 viruses were also found in seven and one case, respectively. The presence of R5^broad and R5X4 phenotypes correlated significantly with a severe decline of the CD4+ T cells (CDC stage 3) or death within 2 years of age. Forty-three percent of the maternal R5 isolates displayed an R5^broad phenotype, however, the presence of the R5^broad virus was not predictive for MTCT of HIV-1. Of interest, while only 1 of 5 mothers with an R5X4 virus transmitted the dualtropic virus, 5 of 6 mothers carrying R5^broad viruses transmitted viruses with a similar broad chimeric coreceptor usage. Thus, the maternal R5^broad phenotype was largely preserved during transmission and could be predictive of the phenotype of the newborn''s viral variant.

Conclusions/Significance

Our results show that R5^broad viruses are not hampered in transmission. When transmitted, immunological failure occurs earlier than in children infected with HIV-1 of R5^narrow phenotype. We believe that this finding is of utmost relevance for therapeutic interventions in pediatric HIV-1 infection.     

Priming of human neutrophils by mycobacterial lipoarabinomannans: role of granule mobilisation.

Lipoarabinomannans (LAMs) from mycobacteria were investigated concerning their effect on human neutrophils. Two types of LAM, the mannose-capped ManLAM from the virulent Mycobacterium tuberculosis H37Rv and the mannose-lacking AraLAM from a rapidly growing mycobacterial strain were used. Neither AraLAM nor ManLAM induced any significant direct activation of the NADPH-oxidase. Both LAMs, however, primed the neutrophils so that subsequent stimulation with the peptide chemoattractants fMet-Leu-Phe (fMLF), Trp-Lys-Tyr-Met-Val-DMet (WKYMVm) and the mammalian lactose-binding lectin galectin-3 resulted in a markedly enhanced oxidative response. The LAM-induced priming was accompanied by an increased exposure of complement receptors 1 and 3 as well as the formyl peptide receptor on the neutrophil surface, suggesting that the enhanced oxidative response could be due to upregulation of receptors on the cell surface as a result of granule mobilisation. Since LAM-primed neutrophils released 65% of the cell content of gelatinase but showed no increased release of vitamin B(12)-binding protein, mobilisation of the gelatinase granules rather than the specific granules is concluded to be responsible for the priming effects. This is in agreement with the subcellular localisation of receptors for fMLF, WKYMVm, as well as galectin-3, which are stored in the secretory vesicles and gelatinase granules. The priming effect appeared very similar to that of Escherichia coli lipopolysaccharide, and since no differences in activity could be detected between AraLAM and ManLAM, we hypothesize that the lipid anchor of the LAM is responsible for the priming effects.     

The dental calculus metabolome in modern and historic samples

Introduction

Dental calculus is a mineralized microbial dental plaque biofilm that forms throughout life by precipitation of salivary calcium salts. Successive cycles of dental plaque growth and calcification make it an unusually well-preserved, long-term record of host-microbial interaction in the archaeological record. Recent studies have confirmed the survival of authentic ancient DNA and proteins within historic and prehistoric dental calculus, making it a promising substrate for investigating oral microbiome evolution via direct measurement and comparison of modern and ancient specimens.

Objective

We present the first comprehensive characterization of the human dental calculus metabolome using a multi-platform approach.

Methods

Ultra performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) quantified 285 metabolites in modern and historic (200 years old) dental calculus, including metabolites of drug and dietary origin. A subset of historic samples was additionally analyzed by high-resolution gas chromatography–MS (GC–MS) and UPLC–MS/MS for further characterization of metabolites and lipids. Metabolite profiles of modern and historic calculus were compared to identify patterns of persistence and loss.

Results

Dipeptides, free amino acids, free nucleotides, and carbohydrates substantially decrease in abundance and ubiquity in archaeological samples, with some exceptions. Lipids generally persist, and saturated and mono-unsaturated medium and long chain fatty acids appear to be well-preserved, while metabolic derivatives related to oxidation and chemical degradation are found at higher levels in archaeological dental calculus than fresh samples.

Conclusions

The results of this study indicate that certain metabolite classes have higher potential for recovery over long time scales and may serve as appropriate targets for oral microbiome evolutionary studies.

     

Optimization of Running Strategies According to the Physiological Parameters for a Two-Runners Model

In order to describe the velocity and the anaerobic energy of two runners competing against each other for middle-distance races, we present a mathematical model relying on an optimal control problem for a system of ordinary differential equations. The model is based on energy conservation and on Newton’s second law: resistive forces, propulsive forces and variations in the maximal oxygen uptake are taken into account. The interaction between the runners provides a minimum for staying 1 m behind one’s competitor. We perform numerical simulations and show how a runner can win a race against someone stronger by taking advantage of staying behind, or how they can improve their personal record by running behind someone else. Our simulations show when it is the best time to overtake, depending on the difference between the athletes. Finally, we compare our numerical results with real data from the men’s 1500 m finals of different competitions.     

Effect of entomopathogenic nematodes on different developmental stages of <Emphasis Type="Italic">Drosophila suzukii</Emphasis> in and outside fruits

Drosophila suzukii Matsumura (Diptera: Drosophilidae) is a harmful invasive fruit pest, which is currently spreading in Europe. Since its arrival in 2008, the spotted wing drosophila has caused major losses in several soft-skinned fruit crops. This critical situation urgently requires efficient practices of residue-free pest control. In the present laboratory study, entomopathogenic nematodes (EPNs) were investigated for their ability to infect larvae and pupae of D. suzukii within directly sprayed fruit, fruit placed on soil, and soil. Steinernema feltiae Filipjev (Rhabditida: Steinernematidae), and Steinernema carpocapsae Weiser (Rhabditida: Steinernematidae) were more efficient at infecting soil-pupating host larvae than Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae) at application rates ranging from 25 to 400 EPN cm^?2. Applied as a soil drench, S. feltiae and S. carpocapsae were able to infect D. suzukii larvae in the soil as well as hidden inside fruit. Direct application of EPNs on the fruit was less successful, although emergence of flies was significantly reduced.